In studies of mammalian genetics, the mouse is the primary research animal, providing models for the analysis of embryonic development and human genetic diseases. Detailed study of the mouse genome, by techniques involving transgenesis and mutagenesis, is leading to the generation of large numbers of new mouse lines world-wide (Nolan et al., Mammalian Genome, 11:500–06, 2000; Thornton et al., Mammalian Genome, 10:987–92, 1999). Although more than 1200 mutations, covering a wide range of phenotypes, have been described in the mouse, this represents just a small fraction (1–2%) of the total number of mouse genes.
Since it is not economically or practically possible to maintain unique mouse stocks by conventional breeding, alternative strategies, such as cryopreservation (the technique of freezing tissues, cells, or other biological materials at very low temperatures, such that cells remain membrane-intact and the materials remain genetically stable and metabollically inert), vitrification, and freezing (without cryoprotection) of embryos or oocytes, have been developed to conserve these valuable genotypes for future study. Such methods have inherent difficulties, however, because large numbers of animals are required, and it is often difficult to obtain sufficient numbers of embryos or oocytes. (See, e.g., Carroll et al., Biol. Reprod., 48:606–12, 1993; Wood et al., Biol. Reprod., 49:489–95, 1993).
The use of spermatozoa frozen with cryoprotection as an alternative means to preserve mouse germplasm has gained scientific interest. However, sperm sensitivity to damage during freezing and thawing has proved to be a major limitation to freezing of mouse spermatozoa (Mazur et al., Cryobiology, 40:187–209, 2000). Within the last few years, cryopreservation and cryostorage of mouse spermatozoa have been achieved with some degree of success, by use of a cyroprotectant in the medium in which the mouse spermatozoa are suspended or stored (Tada et al., J. Reprod. Fertil., 89:511–16, 1990; Nakagata et al., Exp. Anim., 42:317–20, 1993; Sztein et al., Cryobiology, 35:46–52, 1997; Nakagata, Mammalian Genome, 11:572–76, 2000; Sztein et al., Biol. Reprod., 63:1774–80, 2000). Unfortunately, though, spermatozoa of some strains remain very difficult to freeze, e.g., C57BIJ6J (Nakagata et al., Biol. Reprod., 57:1050–55, 1997) and BALB/c (Thornton et al., Mammalian Genome, 10:987–92, 1999).
Conventional techniques for freezing spermatozoa with cryoprotection often utilize physiological suspension media (e.g., to maintain tissues in a viable state) to suspend spermatozoa prior to freezing. Such media contain specific concentrations of substances that are vital for normal tissue function (e.g., sodium, potassium, calcium, chloride, magnesium, bicarbonate, and phosphate ions, as well as glucose and oxygen), and also have appropriate osmotic pressures. A common example of a physiological medium is Ringer's solution, which is used to maintain organs and tissues alive outside of the animal or human body for limited periods. Ringer's solution is an aqueous solution containing sodium chloride, potassium chloride, and calcium chloride, and has an osmotic pressure the same as that of blood serum. There is, however, no single known physiological medium which can support the survival of tissues and organs of all animal species. For cryopreservation applications, cryoprotectants, including sugars, glycerol, dimethylsulfoxide, propylene glycol, ethylene glycol, methanol, 2,3-butanediol, 1,4-butanediol, and dextrans (ranging from 10 to 2000 kDa), are added to the physiological suspension medium (Kundu et al., Reproduction, 123(6):907–13, 2002) in order to maintain cell or tissue viability.
Chelating agents have been used widely in research directed to spermatozoa fertility. In particular, chelating agents have been studied both as fertility inhibitors, e.g., as spermicide additives (Yu et al., Int. J. Androl., 10(6):741–46, 1987), and as fertility promoters, e.g., as enhancers of chromatin decondensation. For example, Rodriguez et al. have demonstrated that the use of chelating agents to bind metal ions that are present in semen results in enhanced chromatin decondensation in ram sperm (Rodriguez et al., Int. J. Androl., 8(2):147–58, 1985). Furthermore, U.S. Pat. No. 5,773,217, issued to L. J. Wangh, discloses use of chromatin-decondensation-enhancing chelating agents to pretreat permeabilized sperm cells prior to sperm-nucleus activation. Nevertheless, chelating agents are also known to exhibit unwanted aneuploidogenic properties that can lead to chromosomal aberrations (Russo et al., Environ. Mol. Mutagen., 19(2):125–31, 1992).
Chelating agents with fertility-promoting properties have also been added to spermatozoa suspension media in non-frozen storage applications. WO 02/24872 describes a nuclear-extraction buffer containing chromatin-decondensation-enhancing chelating agents for washing and storing sperm samples prior to analysis or interaction with other cells or media. However, the addition of chelating agents to an ambient-temperature storage solution, while maintaining the oocyte-penetrating ability of sperm, promotes a high rate of intracellular metabolic activity that may lead to chromatin damage and chromosomal abnormalities (Vishwanath et al., Reprod. Fertil. Dev., 9(3):321–31, 1999). Other studies have utilized chelating agents as an ancillary additive to a physiological medium using trehalose as a cryoprotectant, where the chelating agents prevent cation competition with the cyroprotectant for membrane-binding sites (Aisen et al., Theriogenology, 53(5):1053–61, 2000).
To date, no known studies have investigated the protective properties of chelating agents in connection with spermatozoa that have been frozen without cryoprotection, freeze-dried, or freeze-thawed, or demonstrated the ability of chelating agents to preserve the genetic integrity of spermatozoa undergoing the processes of freezing without cryoprotection, freeze-drying, or freeze-thawing.